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Cyclic adenosine 3',5'-monophosphate negatively regulates clusterin gene expression in Leydig tumor cell lines
O P Pignataro1, Z M Feng, C L Chen
1Population Council, New York, New York 10021.
Abstract:
The clusterin protein and its messenger RNA were identified in many tissues including testis. In this report, we demonstrate the expression of clusterin gene in four Leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540. When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed. A 60-70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in I-10 cells, and no apparent change in LC-540 cells. The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines. The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration. Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA. The suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels. Our observations suggest: 1) cAMP negatively regulates clusterin gene expression in two Leydig tumor cell lines, MA-10 and R2C; 2) The inhibitory effect of cAMP on clusterin gene expression is probably acting through the protein kinase A pathway; and 3) The four Leydig tumor cell lines respond differently to cAMP in the expression of clusterin and side-chain cleavage genes.
Insights
Cyclic AMP (cAMP) significantly suppresses clusterin gene expression in Leydig tumor cells. This inhibitory effect, mediated by protein kinase A, varies across different cell lines and impacts clusterin mRNA levels.
Area of Science:
- Molecular Endocrinology
- Gene Regulation
- Cancer Biology
Background:
- Clusterin is a protein found in various tissues, including the testis.
- Leydig tumor cell lines provide a model to study gene expression in testicular cancer.
Purpose of the Study:
- To investigate the effect of cyclic AMP (cAMP) on clusterin gene expression in Leydig tumor cell lines.
- To determine the mechanism and specificity of cAMP's influence on clusterin mRNA levels.
Main Methods:
- Incubation of four Leydig tumor cell lines (MA-10, I-10, R2C, LC-540) with cAMP-elevating agents (8-bromo-cAMP, (Bu)2cAMP, human CG, forskolin).
- Quantification of clusterin mRNA levels using techniques like Northern blotting or RT-PCR.
- Assessment of other gene expressions (cholesterol side-chain cleavage enzyme, alpha-tubulin) for specificity.
Main Results:
- cAMP treatment led to a significant decrease in clusterin mRNA accumulation in MA-10 and R2C cells (60-70%), with a smaller reduction in I-10 cells (10%) and no change in LC-540 cells.
- The inhibitory effect of cAMP was specific to clusterin, as cholesterol side-chain cleavage enzyme mRNA increased, and alpha-tubulin mRNA remained unchanged.
- The suppression was observed as early as 4 hours and reversed upon removal of cAMP, suggesting a dynamic regulatory process.
- Human chorionic gonadotropin (hCG) and forskolin also suppressed clusterin gene expression, consistent with cAMP-mediated regulation.
Conclusions:
- cAMP negatively regulates clusterin gene expression in MA-10 and R2C Leydig tumor cells.
- The protein kinase A (PKA) pathway is likely involved in mediating the inhibitory effect of cAMP on clusterin.
- Differential responses of Leydig tumor cell lines to cAMP highlight cell-specific regulation of clusterin and steroidogenic genes.